Retro-α-retinol (4,14-retro-retinol) derivatives and uses of retro-α-retinol

ABSTRACT

This invention provides a purified retro-retinoid compound characterized by a molecular mass of about 302 daltons. Also provided by this invention is a method of enhancing the growth of a cell in a vitamin A reduced environment which comprises contacting the cell with an effective growth enhancing amount of a compound having a structure: ##STR1## wherein the configuration of the C6, C8, C10 and C12 double bond independently is Z or E and the absolute configuration at C-14 is independently R or S; wherein R 1  is alkyl, alkyl halide, alcohol, ester, ether, aldehyde, ketone, carboxylic acid, carboxylic ester, acid halide, amide, nitrile, or amine; and wherein R 2  is hydroxyl, halide, alkoxy, ester, alkyl, alcohol, ether, aldehyde, ketone, carboxylic acid, carboxylic ester, nitrile, amine, azide, alkyl halide, acid halide, acid azide, or amide; or wherein R 1  and R 2  are replaced by a 14, 15-oxirane group; and wherein the retro structure is alpha or gamma. Further provided are a method for enhancing transcription of a gene regulated by retinoid in a cell, or a method for enhancing an immune response in a subject.

The invention described herein was made in the course of work underGrant Number CA 499 33 from the United States Government. The UnitedStates Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced to byarabic numeral within parenthesis. Full bibliographic citations forthese references may be found at the end of the specificationimmediately preceding the claims. The disclosures for the publicationsin their entireties are hereby incorporated by reference into thisapplication to more fully describe the state of the art to which thisinvention pertains.

It has long been known that dietary restriction of vitamin A causeswidespread abnormalities in tissue and organ physiology, especially inneonates. The vitamin A deficiency syndrome is characterized bygenerally stunted growth, keratoses of skin and eyes (1) (leading insevere cases to blindness), defective testis development (2) etc., andatrophy of central (i.e., thymus and bursa of Fabrizius) and peripherallymphoid organs. Consequently, immune functions are severely affected.Even in mild cases of vitamin A deficiency, the immune system appears tobe hyporesponsive. In a recent study in southern India (4), the authorsnoted in children suffering from mild vitamin A deprivationsignificantly higher mortality rates in common childhood diseasescompared with children receiving normal dietary levels of vitamin A.Since severity but not susceptibility to infection was correlated withvitamin A deprivation, it is likely that reduced immune functions are afactor.

In the absence of retinol, lymphoblastoid cells (LCL) die within 24 to48 hours. (5) Retinol and retinaldehyde, but not retinoic acid, supportthe growth of LCL in serum-free medium. The same is true for activatedhuman thymocytes. These finding may represent direct correlates to thelagging development of lymphoid organs described by Wolbach and Howe (3)and the in-vivo immune system dysfunction referred to earlier. (4)

Nearly all vertebrate tissues are bathed in a constant supply of vitaminA, and the ubiquitous distribution of cellular retinol-binding protein(CRBP) with its high affinity to retinol suggests that it is inside mostcells as well. Yet the general purpose of retinol, its metabolism andfinal destination, remain for the most part unknown, the well-studiedexample of specialized usage such as vision notwithstanding. Sinceretinal is not known to be incorporated into structural parts of cellsand does not bind to one of the yet analyzed transcription factors withhigh enough affinity, its role is more likely to be found in itsfunction as precursor for derivatives. Use of retinaldehyde in vision isone example, and another that of retinoic acid as a morphogen (6),important for development of limb and brain. When coupled with paralleldiscoveries of retinoic acid receptors (RAR) (7A-7C) within the largersteroid receptor superfamily (8), a sound molecular foundation is given.In this hypothesis, RARs bind to specific response elements in thepromoter regions of genes. Retinoic acid in turn binds to RAR, causingactivation of gene transcription. The universal principle of thisgenetic control has increasingly been highlighted by observations thatmany developmentally important genes from drosophila to man are part ofthe retinoic acid/steroid receptor superfamily. Moreover, for more thantwo dozen "orphan receptors" (9) engaged in control of the generalphysiology of cells, the ligands are not known and are suspected to besmall lipophilic molecules.

In analogy to retinoic acid, other members of the retinoid family mayalso serve as transcriptional activators. This concept has been pursuedin the current invention leading to the discovery of retinoid moleculehitherto unknown in nature, that can activate certain physiologicalprocesses in β lymphocytes. This new compound,14-hydroxy-4,14-retro-retinol (14-hydroxy-retro-α-retinol) may workalong a pathway parallel to the well established retinoic acid pathway,but leading to distinct physiological responses.

SUMMARY OF THE INVENTION

This invention provides a purified retro-retinoid compound characterizedby a molecular mass of about 302 daltons. Also provided by thisinvention is a method of enhancing the growth of a cell in a vitamin Areduced environment which comprises contacting the cell with aneffective growth enhancing amount of a compound having a structure:##STR2## wherein the configuration of the C6, C8, C10 and C12 doublebond independently is Z or E and the absolute configuration at C-14 isindependently R or S; wherein R₁ is alkyl, alkyl halide, alcohol, ester,ether, aldehyde, ketone, carboxylic acid, carboxylic ester, acid halide,amide, nitrile, or amine; and wherein R₂ is hydroxyl, halide, alkoxy,ester, alkyl, alcohol, ether, aldehyde, ketone, carboxylic acid,carboxylic ester, nitrile, amine, azide, alkyl halide, acid halide, acidazide, or amide; or wherein R₁ and R₂ are replaced by a 14, 15-oxiranegroup; and wherein the retro structure is alpha or gamma.

This invention further provides a method for enhancing transcription ofa gene regulated by retinol in a cell which comprises contacting thecell with an effective transcription-enhancing amount of a compoundhaving the structure: ##STR3## wherein the configuration of the C6, C8,C10 and C12 double bond independently is Z or E and the absoluteconfiguration at C-14 is independently R or S; wherein R₁ is alkyl,alkyl halide, alcohol, ester, ether, aldehyde, ketone, carboxylic acid,carboxylic ester, acid halide, amide, nitrile, or amine; and wherein R₂is hydroxyl, halide, alkoxy, ester, alkyl, alcohol, ether, aldehyde,ketone, carboxylic acid, carboxylic ester, nitrile, amine, azide, alkylhalide, acid halide, acid azide, or amide; or wherein R₁ and R₂ arereplaced by a 14, 15-oxirane group; and wherein the retro structure isalpha or gamma.

A method for enhancing an immune response in a subject is also providedby this invention which comprises administering to the subject aneffective immune-enhancing amount of a compound having the structure:##STR4## wherein the configuration of the C6, C8, C10 and C12 doublebond independently is Z or E and the absolute configuration at C-14 isindependently R or S; wherein R₁ is alkyl, alkyl halide, alcohol, ester,ether, aldehyde, ketone, carboxylic acid, carboxylic ester, acid halide,amide, nitrile, or amine; and wherein R₂ is hydroxyl, halide, alkoxy,ester, alkyl, alcohol, ether, aldehyde, ketone, carboxylic acid,carboxylic ester, nitrile, amine, azide, alkyl halide, acid halide, acidazide, or amide; or wherein R₁ and R₂ are replaced by a 14, 15-oxiranegroup; and wherein the retro structure is alpha or gamma.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIG. 1 shows that protein and lipids of conditioned medium aresynergistic in their ability to sustain the growth of β lymphocytes.

4 liters of HB101 medium were conditioned overnight with 400,000 LCL 5/2cells/ml in HB101 medium. The medium proteins were precipitated with 80%ammonium sulfate. The proteins were freeze-dried and delipidated withether/ethanol. 5/2 cells (1000/well) were incubated for 72 hours inHB101 medium with the indicated amounts of delipidated protein andextracted lipids. DNA synthesis was measured by [³ H]-thymidine uptake.The measurements were done in triplicate.

FIG. 2A is the EI mass spectrum of bioactive lipid in human serum.

FIG. 2B is the EI mass spectrum of all-trans retinol from the NationalBureau of Standards Library.

FIG. 3 shows the dose-response curves of different retinoids tostimulate the growth of 5/2 cells in culture.

Washed 5/2 cells (5,000/well) were incubated for 72 hours in HB101medium with the indicated amounts of retinoids. DNA synthesis wasmeasured by [³ H]thymidine uptake. The measurements were done intriplicate. The SDs were <15%.

FIG. 4 shows that retinol but not synthetic retinoic acid analogs enable5/2 cells to grow.

FIG. 4A is the chemical structure of retinoids used.

FIG. 4B is the dose-response curves measured on day 3.

FIG. 4C shows the effect of 3×10⁻⁷ M retinoids measured day 1, 2 and 3.FIG. 4D shows the combination of retinoic acid analogs (3×10⁻⁷ M) withand without 10⁻⁶ M retinol measured on day 3. In FIGS. 4B to 4D, 5/2cells were washed twice and seeded at a concentration of 150,000cells/ml in HB 101 medium. Triplicate samples of 100 μl of cellsuspension were removed daily and pulsed for 6 hours with [³ H]thymidine. Means are shown. The SDs were never >20%.

FIG. 5 shows that retinol deprivation leads to cell death.

5/2 cells were washed once and seeded at a density of 300,000/ml in HB101 medium with and without 10⁻⁶ M retinol. The trypanblue-negative(FIG. 5A) and trypanblue-positive (FIG. 5B) cell number of nine aliquotswas determined every 24 hours. Means +SDs are shown. In a repeatexperiment cells were stained with Wright-Giemsa stain after 40 hours ofculture.

FIG. 5C shows cells without retinol.

FIG. 5D shows that cells with 10⁻⁶ M retinol.

FIG. 6 shows that effect of retinol-deprivation on RNA and DNA contentof 5/2 cells.

5/2 cells were washed and seeded at a density of 50,000/ml in HB 101without (FIG. 6A) and with (FIG. 6B) 10⁻⁶ M retinol. After 48 hours, thecells were stained with acridine orange and 5,000 cells/sample wereanalyzed by flow cytometry. Scattergrams represent distribution of cellswith respect to their DNA and RNA content. 2n corresponds to diploid, 4nto tetraploid DNA content. The boxed dots with very low RNA contentcorrespond to nuclei.

FIG. 7 shows retinol metabolites in 5/2 cells.

5/2 cells (10⁶ cells in 10 ml HB 101 medium) were incubated withall-trans-[³ H]retinol (10 uCe/ml). After 24 hours, retinoids wereextracted from the washed cell pellet and unlabeled marker retinoidswere added. The crude extract was loaded on an analytical reversed-phaseC-18 column. Retinoids were eluted with the shown linear gradient ofwater/methanol/chloroform. The flow rate was 0.5 ml/min. DPM weredetermined with an on line scintillation counter. Reference retinoidswere the all-trans forms of 0: 3,4-didehydroretinoic acid, 1:all-trans-retinoic acid 2: 3,4-didehydroretinol 3: retinol 4: retinyllinoleate 5: retinyl oleate 6: retinyl palmitate. Shaded areacorresponds to P3. Cross-hatched area corresponds to P1

FIG. 8 shows the dose-response curve of P3 and retinol.

P3 was purified as described in the Materials and Methods section of theDetailed Description. 5/2 cells (5,000/well) were incubated for 72 hoursin HB101 medium and refed with the indicated amount of retinoid daily.DNA synthesis was measured by [³ H]-thymidine uptake. P3 is bioactivedown to a concentration of 10-⁸ M (FIG. 8). It is 10 to 15 times morepotent than retinol, but unlike with retinol, cultures have to bereplenished daily with P3. This is due either to chemical instability orto a more rapid metabolic degradation of P3 by the cells.

The use of ³ H retinol bound to fetal calf serum was used as an assay totest for P3 in other cell lines. All 26 mammalian cells tested resultsof 13 cells lines shown in Table 1, radioactivity peak at the positionwhere P3 normally elutes. In the instances tested, the material in thispeak also showed the characteristic UV spectrum of P3.

FIG. 9A shows the absorption spectrum of P3 in methanol, measured on thePerkin-Elmer Model Lambda 4B UV/VIS spectrophotometer. P3 has a λmax at348 nm, a vibronic fine structure at 366, 332,316 and 300 nm, and a weakabsorption at 252 nm. Retinoids show a fine structure in theirabsorption spectra when the molecule adopts a ring/side-chain planargeometry, either imposed by protein/retinoid interaction as in theretinol/CRBP complex or (9A) by a retro-configuration of the double bondsystem as shown in FIG. 9B (9B, 9C). Since P3 maintains its finestructure after the lipid/protein separation, protein/retinoidinteraction are excluded.

FIG. 10 shows circular dichroism spectrum of P3 on the Jasco J-720spectropolarimeter. The CD spectrum of P3 exhibits a positive Cottoneffect and fine structure. This confirms the presence of an assymericcenter. The absolute configuration at C-14 is assigned as R on the basisof the positive Cotton effects associated with respective finestructured UV absorption, i.e., "allylic Hydroxyl effect" (12, 13, 14).

However, since this interpretation is dependent on the perturbation ofthe pentaene absorption at 348 nm by the hydroxyl group, ca. 200 nm(remote from 348 nm), and furthermore, since an additional 15-OH groupis present, the configuration at C-14 needs to be confirmed by ongoingsynthesis.

FIG. 11A shows the low resolution EI mass spectrum of P3, measured onJEOL DX-303 HF.

Low resolution EI/MS of P3 measured on JEOL DX-303 HF m/z 302 (M+ 100),284 (11; M-H₂ O), 271 (23; M- CH₂ OH), 253 (2), 241 (4), 228 (4), 215(6), 197 (6), 187 (9), 173 (10), 159 (15), 147 (17), 133 (15), 121 (23),105 (20). The low resolution mass spectrum indicates the presence of asingle compound with a molecular mass of 302 daltons.

FIG. 11B shows the high resolution EI/MS (matrix PFK). The observed massof 302.2265 (calc. 302.2246) is consistent with an atomic composition ofC₂₀ H₃₀ O₂. This means that P3, which has a retro structure skeleton assuggested by absorption spectroscopy, possesses an additional oxygenatom as compared to its precursor retinol.

FIG. 12 shows proton NMR studies that established that P3 is a14-hydroxy-retro-α-retinol. The NMR spectrum assignment was carried outby decoupling experiments and comparison with literature data given forretro-α-retinylacetate (2).

FIG. 12A shows ¹ H NMR (CD₃ CN, VARIAN vxr-400) §1.30 (S 1-(CH-₃)2),1.50 (t,J 7.5 Hz, 2-CH₂), 1.76a (a, 13-CH3, 1.87a (a, 9-CH3), 1.90b (a,5-CH3), 2.08 (m, 3-CH2), 3.4 and 3.5 (2m, 15-CH2), 4.02 (m, 14-CH), 5.79(t, J 4 Hz, 4-CH), 6.17 (d, J 12 Hz, 12-CH), 6.38 (d, J 12.3, 7-CH),6.42 (d, J 17, 10-CH), 5.56 (dd, J 17, 12 Hz, 11-CH), 6.76 (d, J 12.3,8-CH).

FIG. 12B shows ¹ H NMR of P3 in CD3CN after addition of few drops D20;insert shows 14-CH and 15-CH2 signals in C6D6.

The 5-Me signal at 1.90 ppm, in FIG. 12A is masked by the CH3CNmultiplet at 1.93 ppm; however upon addition of few drops of D₂ O to thesample, the latter signal shifts downfield, unmasking the 5-Me singlet(FIG. 12B). The 9-Me and 13-Me assignments are uncertain and have to beconfirmed by synthesis.

The position of the 6-7 was established as E by comparison of thechemical shifts of 1-(Me (1,30), 5-Me (1.90) and 4-H (5.79) with thosereported for 6-E-retro-α-retinyl acetate (15), 1.28, 1.91, 5.76respectively. The corresponding signals for 6-Z-retro-α-retinyl acetate,are 1.11, 2.06 and 5.63. Furthermore the 6-E configuration of P3 wasconfirmed by observation of NOE (ca. 4%) between 1-(Me)2 and 8-H. Theconfiguration of the 12-13 double bond, is to be confirmed by ongoingsynthesis.

FIG. 13 shows the fluorescent emission spectrum of P3.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a purified retro-retinoid compound characterizedby a molecular mass of about 302 daltons. The purified retro-retinoidcompound of this invention is characterized by a compound having theatomic composition C₂₀ H₃₀ O₂ and having the structure: ##STR5## whereinthe configuration of the C6, C8, C10 and C12 double bond independentlyis Z or E; and R₁ is CH₂ OH and R₂ is OH. However, in the preferredembodiment, the C6 double bond is trans. As used herein, the term"compound" shall mean all isomeric forms of the above compound as wellas all homologs and analogs thereof. This compound may be purified fromnatural sources or chemically synthesized.

This invention also provides a pharmaceutical composition whichcomprises the purified retro-retinoid compound described hereinabove oralternatively, a synthetic product of the compound, and apharmaceutically acceptable carrier. As used herein, the term"pharmaceutically acceptable carrier" encompasses any of the standardpharmaceutically carriers, such as a phosphate buffered saline solution,water, and emulsions such as an oil/water emulsion, and various types ofwetting agents. In the preferred embodiment of the invention, thepharmaceutically acceptable carrier also comprises specific bindingproteins, which may be, but are not limited to retinol binding protein(RBP), transthyretin (TTR), the complex formed by RBP and TTR, andalbumin. Most specifically, the complex composition shall have a ratioof 4:1:1 with respect to RBP, TTR and the retro-retinol compound and aconcentration of about 10 to about 100 μg/ml. Albumin is at aconcentration of 1 microgram per milliliter.

This invention also provides a method for obtaining the purifiedretro-retinoid compound described hereinabove which comprises growing asuitable cell line under suitable conditions, contacting the grown cellswith 10⁻⁵ M all-trans retinol, extracting the cell pellet or the culturefluid with organic solvents such as, but not limited to, butanol,acetonitrile ethyl ether, chloroform, methylene chloride, separating theorganic phase from the cell pellet or culture fluid, and isolating theretro-retinoid compound by HPLC column chromatography, wherein theretro-retinoid compound elutes on a C-18 column at 83% methanol/17%water.

In the preferred embodiment of this invention, the suitable cell line isa HeLa cell line, although other mammalian and avian cell lines, such aslymphoid cells, fibroblasts, myeloid, neuroblastoma, teratoma, hepatomaand breast carcinoma can also be utilized in this method. With respectto the malignant or transformed cell lines listed above, the "normal" ornon-transformed or malignant line also is useful in this method. Cellshould be grown in a nutrient medium such as Eagles modified mediumcontaining 10% bovine serum. All-trans retinol is then added. The cellsare then separated from the liquid medium and washed with a neutralsolution such as phosphate buffered saline (PBS). Cells should then beresuspended in the neutral solution and an organic solvent such asbutanol/acetonitrile is added. The cells should be vortexed andsaturated K₂ HPO₄ is added. The cells are again vortexed and the organicphase should be separated. The compound is then isolated by a runthrough C-18 column preequilibrated with water, and run through with agradient of methanol/water to yield the compound at 83% methanol/17%water.

Also provided by this invention is the compound described above as wellas all isomeric forms of the above compound, and homologs and analogsthereof. This compound may be administered in a pharmaceuticallyacceptable carrier. As used herein, the term "pharmaceuticallyacceptable carrier" encompasses any of the standard pharmaceuticallycarriers, such as a phosphate buffered saline solution, water, andemulsions such as an oil/water emulsion, and various types of wettingagents. In the preferred embodiment of the invention, thepharmaceutically acceptable carrier also comprises specific bindingproteins, which may be, but are not limited to retinol binding protein(RBP), transthyrein (TTR), the complex formed by RBP and TTR, andalbumin. Most specifically, the complex composition shall have a ratioof 4:1:1 with respect to RBP, TTR and the retro-retinol compound and aconcentration of about 10 to 100 μg/ml. Albumin is at a concentration of1 microgram per milliliter.

This invention further provides a method of enhancing the growth of acell in a vitamin A reduced environment which comprises contacting acell with an effective growth enhancing amount of a compound having thestructure: ##STR6## wherein the configuration of the C6, C8, C10 and C12double bond independently is Z or E and the absolute configuration atC-14 is independently R or S; wherein R₁ is alkyl, alkyl halide,alcohol, ester, ether, aldehyde, ketone, carboxylic acid, carboxylicester, acid halide, amide, nitrile, or amine; and wherein R₂ ishydroxyl, halide, alkoxy, ester, alkyl, alcohol, ether, aldehyde,ketone, carboxylic acid, carboxylic ester, nitrile, amine, azide, alkylhalide, acid halide, acid azide, or amide; or wherein R₁ and R₂ arereplaced by a 14, 15-oxirane group; and wherein the retro structure isalpha or gamma.

As used herein, the term "enhancing the growth of a cell" means anincrease of its proliferation, i.e., an increase in the cell number as aconsequence of cell division. In addition, the term "vitamin A reducedenvironment" shall mean culture medium containing less than about 10⁻⁷ Mvitamin A.

The method may be practiced in vitro or in vivo. If the method ispracticed in vitro, contacting may be effected by incubating the cellswith the compound. The concentration of the compound is theconcentration which is effective to enhance the growth of the cell asdescribed in FIG. 8. Therefore, the effective amount is varied with thetype of cell.

Another factor in determining the effective amount of the compound isthe degree of vitamin A deficiency in the environment. Thus, theeffective concentration of each compound will also vary with the degreeof vitamin A deficiency within the cell and the amount of compensationwhich is to be provided by the compound.

The method of the present invention is also intended for the treatmentof animals, e.g. mammals, including human patients. When the compound isto be administered in vivo, it is intended that the compound beadministered as a composition comprising the compound in apharmaceutically acceptable carrier.

Methods of the administration to animals are well known to those ofskill in the art and include, but are not limited to, administration,intravenously or parenterally. Administration of the composition will bein a dosage such that the compound enhances the growth of the cell to beeffected. Administration may be effected continuously or intermittentlysuch that the amount of the composition in the patient is effective toenhance the growth of the target cell to be effected.

In the preferred embodiment of this invention, R₁ is CH₂ OH and R₂ is--OH, and the C6 double bond is trans. In addition, administration ofthe compound is effected continuously.

This invention also provides a method for enhancing transcription of agene regulated by retinoids in any cell which comprises contacting thecell with an effective transcription enhancing amount of a compoundhaving the structure: ##STR7## wherein the configuration of the C6, C8,C10 and C12 double bond independently is Z or E and the absoluteconfiguration at C-14 is independently R or S; wherein R₁ is alkyl,alkyl halide, alcohol, ester, ether, aldehyde, ketone, carboxylic acid,carboxylic ester, acid halide, amide, nitrile, or amine; and wherein R₂is hydroxyl, halide, alkoxy, ester, alkyl, alcohol, ether, aldehyde,ketone, carboxylic acid, carboxylic ester, nitrile, amine, azide, alkylhalide, acid halide, acid azide, or amide; or wherein R₁ and R₂ arereplaced by a 14, 15-oxirane group; and wherein the retro structure isalpha or gamma.

As used herein, the term "enhancing transcription of a gene" is definedas the accelerated production of messenger RNA in cells. C-fos andCD-38, are two examples of genes which are regulated by retinol andtherefore, whose transcription may be enhanced by the use of the claimedmethod.

As used herein, the term contacting, is to mean contacting in vitro orin vivo. Methods of in vitro and in vivo contacting are describedhereinabove. The effective amount of a compound is the amount whichenhances transcription of certain genes in the cell and will vary withthe type of cell as well as the gene to be regulated. Methods ofdetermining the effective amount are well known to those of skill in theart.

This invention also provides a method for enhancing an immune responsewhich comprises administering to the subject an effectiveimmune-enhancing amount of a compound having the structure: ##STR8##wherein the configuration of the C6, C8, C10 and C12 double bondindependently is Z or E and the absolute configuration at C-14 isindependently R or S; wherein R₁ is alkyl, alkyl halide, alcohol, ester,ether, aldehyde, ketone, carboxylic acid, carboxylic ester, acid halide,amide, nitrile, or amine; and wherein R₂ is hydroxyl, halide, alkoxy,ester, alkyl, alcohol, ether, aldehyde, ketone, carboxylic acid,carboxylic ester, nitrile, amine, azide, alkyl halide, acid halide, acidazide, or amide; or wherein R₁ and R₂ are replaced by a 14, 15-oxiranegroup; and wherein the retro structure is alpha or gamma and apharmaceutically acceptable carrier. In the preferred embodiment of thisinvention, R₁ is CH₂ OH and R₂ is --OH and the C6 position is trans.This method is effective for enhancing the subject's cellular immuneresponse as well as the subject's humoral immune response. As usedherein, the definition of the terms "cellular immune response" and"humoral immune response" are known to those of skill in the art. Forthe purposes of this invention, the subject may be, but is not limitedto, an animal, such as a mammal, or a human patient.

It is contemplated that this invention is to be practiced in vivo.Accordingly, an effective amount is an amount which is effected toenhance the immune response of the subject. Accordingly, the effectiveamount will vary with the subject being treated, as well as thecondition to be treated. For the purposes of this invention, the methodsof administration are to include, but are not limited to, administrationintravenously or parenterally.

This invention is illustrated in the Materials and Methods section whichfollows. This section is set forth to aid in an understanding of theinvention but is not intended to, and should not be construed to, limitin any way the invention as set forth in the claims which follow.

MATERIALS AND METHODS Retinoids

Ro-10-1670 (Etretine, Ro 13-7410 (TTNPB), Ro 40-6085 (AM 80), and3,4,-didehydroretinol were generous gifts of Hoffman-LaRoche, Inc.Nutley, N.J. 3,4-didehydoretinol was oxidized to 3,4-didehydroretinaland 3,4-didehydroretinoic acid according to the procedure of Mayer etal. (3). Retinyl esters were a gift of Dr. W. Blaner, ColumbiaUniversity, N.Y. All other unlabeled retinoids used were purchased fromSigma Chemical Co. (St. Louis, Mo.). [³ H]retinol was purchased fromAmersham, Arlington Heights, Ill. and was >98% pure according to HPLCanalysis. The retinoids were dissolved in methanol/chloroform (3:1)(vol/vol) at a concentration of 3×10⁻² M with 10⁻⁴ M butylatedhydroxytoluene (BHT) (Sigma Chemical Co.) added and stored in the darkat -20° C. in a nitrogen atmosphere. Immediately before bioassays, thestock solutions were diluted in serum-free medium.

Cell Lines

The human EBV-transformed B-cell line 5/2 was established from theperipheral blood of a healthy donor. The cell line was grown in RPMI1640 supplemented with 8% fetal calf serum, L-glutamine (2 mM), andantibiotics. The cell line was tested regularly for mycoplasmainfections and was consistently negative.

Synthesis of Retroretinol

Retroretinol is prepared by treatment of retinal enolacetate with NaBH₄,and can be converted to retroretinyl acetate by acetylation ofretroretinol (16). Retroretinyl acetate has also been prepared bytreatment of retinyl acetate with aqueous HBr (17). Retrovitamin Amethyl ether was synthesized in 1952 (18). Derivations of the above maybe synthesized by methods well known to those of skill in the art.##STR9##

Chemical Synthesis of P3

Compound #2 (below) is prepared from 2,6,6-trimethylcyclohexenone 1according to methods well known by those of skill in the art (19).Intermediate #3 is synthesized from commercially available (R) or (S)glyceraldehyde. ##STR10##

Cell Proliferation Assay

The assay system is a modification of the assay developed by Blazar etal. (10). Specifically, cells taken from their exponential growth phasewere washed twice and seeded with or without retinoids at graded cellconcentrations in serum-free HB 101 medium (Hana Biologics, Berkeley,Calif.) or in FCS containing RPMI medium. Assays were done in 96 wellmicrotiter plates in a final volume of 200 μl/well or in 25 cm² tissueculture flasks. The cells in the microtiter plates were cultured for 72hours and cell growth was determined by labeling for the last 16 hourswith 0.8 μCi/well of [³ H]thymidine (sp. act. 6.7 Ci/mmol). Growth inthe culture flasks was determined in three aliquots of 100 μl taken at24 hours intervals and cultured in the presence of 0.8 μCi of [³H]thymidine for an additional 6 hour period. To determine the viablecell number, nine aliquots per time point were differentially counted ina Neubauer chamber in the presence of trypan blue.

Purification of P3

HeLa cells were grown in spinner flasks in Eagles modified mediumcontaining 10% bovine serum to a density of 7×10⁵ cells/mi. 10⁻⁵ Mall-trans retinol (Sigma) was added 16 hours before harvesting thecells. The cells were spun down and washed with phosphate bufferedsaline (PBS). 12 ml of packed cells were resuspended in 24 ml PBS and9.6 ml-butanol/acetonitrile 1:1 (v/v) was added. After vortexing for oneminute, 7.2 ml of a saturated K₂ HPO₄ was added. The mixture wasvortexed again for one minute, and the organic phase were separated bycentrifugation at 8000 rpm for 10 minutes.

The organic phase was diluted with an equal volume of water and loadedon a preparative C-18 HPLC column (Vydac) which was preequilibrated withwater. The column was eluted with a gradient of water to methanol. P3eluted at 83% methanol/17% water as determined by the characteristicabsorption spectrum in the photodiode array detector. The P3-containingfraction was rechromatographed on a semipreparative C₁₈ column (Vydac)using as eluant a linear gradient of water to acetonitrile. P3 eluted at25% water, 75% acetonitrile.

To concentrate P3, the purified material from the semipreparative C18column was loaded on an analytical C4 column (VYDAC) and eluted with alinear gradient water to methanol. P3 eluted at 28% water, 72% methanol.10¹¹ HeLa cells yielded 25 optical units of P3 at 348 nm. This materialwas analyzed by NMR.

RNA and DNA Staining

To analyze cell progression through the cell cycle, cells were stainedwith acridine orange (Polysciences Inc., Warrington, Pa.). In brief, 0.4ml of acid detergent (0.1% Triton X-100; 0.08N HCl; 0.15M NaCl) wasadded to 0.2 ml of the cell suspension. Thirty seconds later, 1.2 ml ofacridine orange staining solution (6.0 ug/ml acridine orange, 10⁻³ MEDTA, 0.15M NaCl, 0.1 M citrate-phosphate buffer at pH 6) was added toeach sample. Cells were measured immediately using a FC200 flowcytometer (Ortho Diagnostics, Westwood, Mass.) as described (17,18). Thered (600 to 640 nm) and green (515 to 575 nm) luminescence emissionsfrom each cell were optically separated, measured by separatephotomultipliers, and the data collected and stored in a Compaq Deskpro386 computer. The number of cells in G₁, S and G₂ +M cell cyclecompartments were calculated using interactive computer programs.

RESULTS

Lymphoblastoid 5/2 cells grown in the presence of 10⁻⁶ M retinol werespiked with ³ H-labeled retinol. After 16 hours the cell pellet wasdelipidated according to the method of McLean et al. and separated on areversed phase C-18 HPLC column (FIG. 7). By comigration with standards,13-cis retinol, all-trans retinol, and several retinyl esters wereidentified. Retinoic acid, 3,4-didehydroretinol acid as well as3,4-didehydroretinol were not detectable. Three peaks, P1, P2 and P3,could not be immediately identified. One peak eluting at 36-39 minutes(corresponding 83% methanol 7% water) called P3, was unusual becauseunlike P1 eluting at 31-34 minutes, its relative amount increased whenthe retinol concentration was lowered or when retinol was given to thecells bound to RBP. P3 was tested in the B-cell growth assay and foundit was active and capable of replacing retinol (FIG. 8). Since HeLacells contain P3, these cells were also used. 50 ml of packed HeLa cells(10¹⁰ cells) yielded 25 OD_(348nm) units of pure P3 after the followingsequence of steps: 1. Growth in the presence of 10⁻⁵ M retinol; 2.Extraction of cell pellet according to McClean et al. (11); 3.Preparative C-18 HPLC column eluted with a water/methanol gradient; 4.semipreparative C₁₈ HPLC column eluted with a water/acetonitrilegradient; 5. analytical C₄ column eluted with a water/methanol gradient.P3 is unstable in chloroform and does not survive an acid extractionstep. P3 displayed an absorption with a fine structure and absorptionmaxima at 326.

An EI mass spectrum of P3 was obtained. This spectrum (FIG. 11B)indicated the presence of a compound with a molecular mass of 302daltons. High resolution mass spectroscopy showed a mass of 302.2265,which is consistent with an atomic composition of C₂₀ H₃₀ O₂. This meansthat P3 not only has a retro structure but an additional oxygen atom ascompared with its precursor retinol.

Proton NMR confirmed the features of 14-hydroxy-retro-α-retinolstructure. P3 is bioactive down to a concentration of 10⁻⁸ M. It is 10to 15 times more potent than retinol on a molar basis, but unlineretinol, cultures have to be replenished daily with P3. This is dueeither to chemical instability or to a more rapid metabolic degradationof P3 by the cells.

The use of ³ H retinol bound to fetal calf serum was used as an assay totest for P3 in other cell lines. All 26 mammalian cells tested resultsof 13 cells lines shown in Table 1, radioactivity peak at the positionwhere P3 normally elutes. In the instances tested, the material in thispeak also showed the characteristic UV spectrum of P3.

REFERENCES

1.-3. Wolbach, S. B. et al. J. Exp. Med. 42: 753 (1925).

4. Rahmathullah, L, et al. N. Eng. J. Med. 323:929-935 (1990).

5. Buck, J. et al. J. Exp. Med. 171:1613-1624 (1990).

6. Slack, J. M. Nature (London) 327:553-554 (1987).

7A. Petkovich, M. et al. Nature (London) 330: 444-450 (1987).

7B. Giguere, V. et al. Nature (London) 330:624-629 (1987).

7C. Mangelsdorf, D. J. et al. Nature (London) 345:224-229 (1990).

8. Evans, R. M. Science 240:889-895 (1988).

9A. McDonald, P. N. and Ong, D. E. J. Biol. Chem 262:10550 (1987).

9B. T. Schreckenbach, B. Walckhoff, D. Oesterhelt, Eur. J. Biochem.,76:499-511 (1977).

9C. K. Reppe in "Houben-Weyl, Methoden der Organischen Chemie",Thieme-Verlag Stuttgart (E. Muller Ed.), Vol. V 1d:7-31 (1970).

10. Blazar, B. A. et al. Cancer Res. 43:4562 (1983).

11. McLean, et al. Clin. Chem. 28:693-696 (1982).

12. N. C. Gonnella, K. Nakanishi, V. S. Martin, K. B. Sharpless, J. Am.Chem. Soc., 104:3775-3776 (1982).

13. S. Natori, in "Natural Products Chemistry", K. Kakanishi et al.,Eds. Vol. I, Kodansha Ltd., Tokyo, Academic Press, Inc. New York, N.Y.,pp. 30-32 (1974).

14. A. F. Beecham, Tetrahedron, Vol. 27:5207 (1971).

15. W. Vetter, et al., in "Carotenoids", O. Isler Ed. Berkhauser VerlagBasel, 204-243 (1971).

16. L. Gosswein, 1976, Diplomarbeit. Univ. of Wurzburg.

17. R. H. Beutel et al., J. Am. Chem. Soc. 77:5166-5167 (1955).

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                  TABLE I                                                         ______________________________________                                        Retinal metabolism of 13 selected cell lines                                  % of taken up retinol                                                         Cell type                   Retinol                                                                             Ester                                       cts.       Line     P3(%)   (%)   (%)    Total                                ______________________________________                                        Burkitt's                                                                     lymphoma-human                                                                ×10.sup.5                                                                          Raji 4°                                                                         0       99    0      2.5                                  ×10.sup.5                                                                          Raji 37°                                                                        3       79    14     4.0                                  Lymphoblastoid                                                                           5/2      4       67    24     5.7                                  cells-human                                                                   Lymphoblastoid                                                                           Ket      3       52    41     4.6                                  cells-human                                                                   ×10.sup.5                                                               Leukemia (ALL)                                                                           SKL3     2       71    24     1.6                                  ×10.sup.5                                                               human                                                                         Leukemia (ALL)                                                                           RPMI     2       58    38     1.2                                  ×10.sup.5                                                               human                                                                         Leukemia, pre-B                                                                          SLA      3       85    9      6.1                                  ×10.sup.5                                                               mouse                                                                         Leukemia, T-Cell                                                                         EL-4     5       40    44     2.8                                  ×10.sup.5                                                               mouse                                                                         Leukemia, T-Cell                                                                         ERLD     9       63    20     2.8                                  ×10.sup.5                                                               mouse                                                                         Monocytic  P388D.1  0.5     71    26     1.4                                  ×10.sup.5                                                               leukemia                                                                      mouse                                                                         B-cell hybridoma                                                                         SK3886   3       92    2      4.7                                  ×10.sup.5                                                               Cervical   Hela     3       71    20     1.0                                  ×10.sup.5                                                               carcinoma-human                                                               Teratocarcinoma                                                                          N-tera2  0.4     0.7   97     13.5                                 ×10.sup.5                                                               human                                                                         Neuroblastoma                                                                            SK-N-SH  0.9     56    25     2.0                                  ×10.sup.5                                                               human                                                                         ______________________________________                                         Legend: 2.5 ×  10.sup.6 cells were grown overnight in 5 ml RPMI/10%     FBS. The FBS was preincubated at room temperature with 14.4 uCi [.sup.3 H     retinol (NEN) for 4 h. The cell pellet was washed twice with PBS. The         retinoid were extracted according to MClean et al. 90 and separated on an     analytical c.sub.18 reverse phase column according to FIG. 7. The DPM wer     determined using an online scintillation counter.                        

What is claimed is:
 1. A homogeneous retro-retinoid compound having the retrostructure: ##STR11## where the configuration of the double bond at C6, C8, C10 and C12 is independently Z or E; andwherein the retro structure is (α) or (γ).
 2. The homogeneous retro-retinoid compound of claim 1, wherein the double bond at C6 is E.
 3. A pharmaceutical composition which comprises the homogeneous retro-retinoid compound of claim 1 and a pharmaceutically acceptable carrier.
 4. A method for obtaining the homogenous retro-retinoid compound of claim 1 which comprises:a) growing HeLa cells in Eagles modified medium containing 10% bovine serum to a density of 7×10⁵ cells/ml; b) adding 10⁻⁵ M all-trans retinol to the growing cells 16 hours before harvesting the cells; c) recovering the cells; d) resuspending the cells so recovered in phosphate buffered saline; e) adding butanol/acetonitrile 1:1 (v/v) to the resuspended cells; f) adding saturated K₂ HPO₄ to the resuspended cells from step (e); g) separately recovering the organic phase from step (f); and h) purifying the retro-retinoid compound from the organic phase by HPLC chromatography on a C-18 column, the compound eluting at 83% methanol/17% water when eluted with a gradient of water to methanol, followed by HPLC chromatography on a second C-18 column, the compound eluting at 25% water/75% acetonitrile when eluted with a linear gradient of water to acetonitrile. 